Protease nexin-I(PN-I) is representative of a class of cell-secreted protein inhibitors collectively referred to as the Protease Nexins(PN's). It has a molecular weight of 43,000 Daltons, and is related to, but distinct from the plasma-borne protease inhibitors, heparin cofactor-II(HC-II) and antithrombin-III(AT-III). PN-I forms covalently-linked complexes with thrombin, plasmin, urokinase and trypsin, and mediates their inactivation, binding, internalization and degradation by cells. Thus, PN-I is likely to be a significant regulatory element in a number of important biological processes where these proteases are involved. The interaction of PN-I with cells will be examined. The receptor that mediates the binding and internalization of PN-I:protease complexes will be characterized at the molecular level, purified, and monoclonal antibodies against it will be generated. The molecular characterization will include chemical crosslinking of 125I-Protease:PN-I complexes to receptors on human fibroblasts. The receptor will be purified from human placental membranes using standard biochemical approaches. Either partially purified or homogeneous receptor will be used to generate monoclonal antibodies. These antibodies will be used to study the dynamics and regulation of the PN-I receptor. This will largely be accomplished by employing a combination of metabolic labeling with 35S-methionine followed by immunoprecipitation. PN-I also interacts with cells via cell-surface and extracellular matrix glycosaminoglycans. This leads to an acceleration of linkage formation between PN-I and thrombin on fibroblasts. The heparin binding domain of PN-I will be identified, purified and sequenced using a combination of proteolytic digestion followed by separation on reverse-phase high performance liquid chromatography. Peptides of 25 amino acid or less will be synthesized that overlap within the identified heparin binding domain of PN-I and antibodies will be raised against them. These antibodies will be used to verify the physiological relevance of the identified heparin binding domain by assessing their ability to block heparin binding and/or augment protease inhibitory activity.